Abstract

Lung cancer is the leading cause of cancer-related deaths, and it is estimated that 215,000 new cases of lung cancer will be diagnosed and 160,000 lung cancer-related deaths occur in 2008 in the US alone. Cigarette smoking and exposure to various environmental pollutants are the leading causes of pulmonary morbidity and mortality. After these environmental insults, an inflammatory response is generated that activates macrophages to release cytokines in the lungs for protection. These secreted cytokines have both anti- or pro-tumorigenic action within the tumor microenvironment. Accordingly, we need to identify the signals which eventually induce the biological responses such as proliferation, angiogenesis, invasion, migration and metastasis. TNF-\#945; and IFN-\#947; are two cytokines which play an important role in the lung tumor microenvironment to affect the lung cancer progression. Therefore the aim of the present study was to determine which signal transduction pathways are activated by these cytokines in lung tumor cells. We used the tumor-derived mouse lung epithelial LM2 cells, which harbor a Kras mutation in codon 61. Cells were serum-deprived for 24 h, and then treated with a mixture of cytokines TNF-\#945; (10 ng/ml) and IFN-\#947; (15 ng/ml) for 15 min to 24 h. When cell lysates were analyzed by immunoblot analyses, there was significantly increased phosphorylation of Stat3 after 15 min; Stat1 and JNK1/2 after 3h and Erk1/2 after 12h, which lasted through the 24 h time point. AKT phosphorylation and HIF-1\#945; expression also increased after 15 min of cytokine mixture exposure, and remained elevated up to 1h. We also observed an increased expression of COX2 and iNOS after 12 h. Pre-treatment of cells with PD98059 (MEK1/2 inhibitor), 2 h prior to cytokine treatment, inhibited cytokine-induced activation of Erk1/2 and strongly decreased the induction of both COX2 and iNOS. Pre-treatment with JAK1 and JAK2 inhibitors also decreased cytokine-induced iNOS expression, but increased COX2, suggesting that another approach is necessary to establish the involvement of Stats in COX2 induction that is independent of chemical inhibitors. Together, these results indicate a role of both Stats and Erk1/2 in cytokine-induction of pro-angiogenic molecules COX2 and iNOS in LM2 cells, and that COX2 and iNOS may be important molecules for preventive and therapeutic studies aimed at targeting cytokines secreted within the lung tumor microenvironment.